A CONTRIBUTION TO THE SYSTEMATICS
OF THE REPTILIAN MALARIA PARASITES,
FAMILY PLASMODIIDAE
(APICOMPLEXA: HAEMOSPORORINA)

SAM ROUNTREE TELFORD, Jr.

UNIVERSITY OF FLORIDA

GAINESVILLE

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A CONTRIBUTION TO THE SYSTEMATICS OF THE
REPTILIAN MALARIA PARASITES, FAMILY
PLASMODIIDAE (APICOMPLEXA: HAEMOSPORORINA).

Sam Rountree Telford, Jr.*

ABSTRACT

The malaria parasites of reptiles, represented by over 80 known species, belong to three
genera of the Plasmodiidae: Plasmodium, Fallisia, and Saurocytozoon. Plasmodium, containing
most of the species, is comprised of seven subgenera: Sauramoeba, Carinamoeba, Lacertamoeba,
Paraplasmodium, Asiamoeba, Garnia, and Ophidiella. Of these, Lacertamoeba, Paraplasmodium,
and Asiamoeba are new subgenera. The subgenera are defined on the basis of morphometric
relationships of the pigmented species, by the absence of pigment (Garia), or by their presence
in ophidian hosts (Ophidiella). The pigmented species with schizonts and gametocytes of similar
size are divided into three groups with little or no morphometric overlap: Sauramoeba, with very
large gametocytes and schizonts, which undergo 4 to 7 nuclear divisions in the erythrocytic phase
of the life cycle; Carinamoeba, with very small gametocytes and schizonts, that have 2 or 3
divisions; and Lacertamoeba, with medium-sized gametocytes and schizonts, which undergo 3 to 5
nuclear divisions. Lacertamoeba species that overlap morphometrically with Carinamoeba can be
distinguished from the latter by their larger range of merozoite numbers. Two species groups
have schizonts and gametocytes of dissimilar size: Paraplasmodium has large gametocytes but
schizonts of only medium size, while Asiamoeba has gametocytes 4 to 15 times the schizont size.
Paraplasmodium is further distinguished by containing the only species known to undergo normal
sporogony in a psychodid fly (Lutzomyia), and by its capacity to produce exoerythrocytic
schizonts in both fixed and wandering host cells. Fallisia is characterized by having both asexual
and sexual cycles in non-erythrocytic blood cells, while the asexual stages of Saurocytozoon appear
to be transitory in circulating lymphocytes, disappearing when gametocytes, largely confined to
lymphocytes, become evident.

* The author is an Adjunct Curator at the Florida Museum of Natural History, University of Florida, Gainesville FL 32611.

The malarial parasites of lizards were placed by Garnham (1966) into two
subgenera of Plasmodium, Carinamoeba and Sauramoeba, which were
distinguished by size of erythrocytic schizonts alone. The only definition of the
two groups was that Sauramoeba had "large" schizonts while those of
Carinamoeba were "small." This did not provide a clear criterion for
separation of the species, but given the lack of precision characteristic of most
taxonomic descriptions of saurian malarias up to that time (Telford, 1973), this
was perhaps the only separation possible. Telford and Ball (1969) supposedly
quoted Garnham as limiting Carinamnoeba to those forms with a "maximum of
12 merozoites," citing his comment on page 819 as authority for this limitation.
Viewed in retrospect, this passage probably referred to Plasmodium wenyoni
Garnham, 1966 of Brazilian snakes as having such a maximum merozoite count
rather than Carinamoeba, but it is ambiguous, for earlier (p. 818) 12-14 nuclei
were stated to be produced by P. wenyoni. Ophidiella was proposed as a
subgenus by Garnham to distinguish the poorly known Plasmodium parasites
of snakes from those of saurians.
Twenty-three saurian Plasmodium species were recognized by Garnham
when he erected the subgenera. Two others, Plasmodium brumpti Pelaez and
Perez-Reyes, 1952 and Plasmodium beltrani Pelaez and Perez-Reyes, 1952
were omitted, while description of another Mexican species, Plasmodium
josephinae Pelaez, 1967 had not appeared. Since 1966, 34 additional species
and subspecies of haemosporidians here considered to be Plasmodium have
been described from Neotropical lizards, two from North America, four more
from Asia, one from Australia, and eleven from Africa. Descriptions of a
dozen more are in preparation. The variety of species found in the last twenty
years demands subgeneric reclassification into more precisely defined groups if
possible, which reflect their common characteristics but which do not depart
further than necessary from the basic scheme of classification provided by
Garnham (1966) for mammalian and avian species. It was perhaps inevitable
that the discovery of so many new species produced some which do not easily
fit the clear-cut definitions of haemosporidian families and genera provided by
Garnham, so readily accepted at that time.
The classical definition of the Plasmodiidae, as given by Garnham (1966)
follows: Plasmodiidae Mesnil, 1903. Parasites belonging to this family have a
sexual phase in the mosquito and asexual cycles in tissue and blood cells of the
vertebrate host; gametocytes are produced and are confined to mature
erythrocytes. Malaria pigment is present in certain stages of the parasite."
Garnham recognized a single genus within the Plasmodiidae; the definition of
Plasmodium, therefore, is that of the family. The critical characters of this
definition relate to the mosquito host, asexual cycles in both tissue and blood

BULLETIN FLORIDA STATE MUSEUM VOL. 34(2)

cells, the production of gametocytes and their restriction to erythrocytes, and
the presence of malaria pigment (hemozoin). Neither type of host cell nor
production of a visible metabolic residue, in my opinion, represents a
biologically or evolutionarily significant characters for definition of higher taxa.
Both Ayala (1977) and Levine (1985) have expressed a similar viewpoint.
In 1969 and 1970 I described three saurian malarias from Panama which
did not show visible pigment: Plasmodium balli, Plasmodium gonatodi, and
Plasmodium morulum. Although P. balli was reported as having pigment,
"minute black dots" or "mass larger than a single merozoite" (Telford, 1969)
this was not confirmed as hemozoin under polarized light, and re-examination
of the type slides on several occasions since has not confirmed its presence. I
can only conclude that I was mistaken in describing the occasional presence of
pigment in this species. In 1973 I reported the presence of pigment in some
gametocytes ofP. morulum. This was confirmed under polarized light, and it is
difficult to consider this to be error. The sample in question was from an
experimental infection in at least the fourth passage of the original isolation of
P. morulum, which makes it most unlikely that another, pigmented species was
also present but undetected that long. Plasmodium azurophilum from the
Caribbean is typically unpigmented (Telford, 1975), but pigment was observed
in 0.2 percent of the gametocytes (N= 800+), and confirmed under polarized
light as hemozoin. In the other unpigmented species which I have studied--
Plasmodium scorzai and Plasmodium lainsoni (Telford, 1978) and P. gonatodi
(Telford, 1970)--1 have never detected pigment. Although Plasmodium beebei
(Telford, 1978) appears closely related to P. gonatodi by having very similar,
bizarrely shaped "prematuration" gametocytes, it is pigmented. Another
normally pigmented species, Plasmodium tropiduri panamense, is often seen
without pigment in gametocytes and seldom shows pigment in schizonts. Here,
the presence of pigment is correlated with maturity of host cell: those found in
erythrocytes are more frequently pigmented than are those which occupy
immature red blood cells (Telford, 1979). Plasmodium telfordi, was described
as unpigmented by Lainson, Landau, and Shaw (1973). I found
morphologically indistinguishable parasites in the same host, Ameiva ameiva
from Guyana, which were frequently pigmented (Telford, 1973). Later, in
Venezuela (1980), I found apparent P. telfordi infections in A. ameiva to be
consistently pigmented--yet found a single infection that was almost entirely
unpigmented. The host cells for this latter infection were mostly
proerythrocytes.
From the above observations I have concluded that the production of
visible pigment by some saurian malarial species is a variable character,
correlated with parasitizing immature erythrocytes, and is therefore unreliable
as a single criterion for subgeneric or generic classification. Other species,
however, never (or very rarely) produce hemozoin, despite occupying
erythrocytes, and these may form a natural group taxonomically. Lainson,

TELFORD: CONTRIBUTIONTO PLASMODIID SYSTEMATICS

Landau and Shaw (1971) erected a separate family and genus, Garniidae and
Gamia for four species which lacked pigment visible under light microscopy.
Although I did not accept their taxa at the levels proposed (Telford, 1973), I
commented that Gamia might be valid as a subgenus of Plasmodium, and it
will be used thus in the classification presented below.
In 1970 I reported the presence of gametocytes and schizonts in
thrombocytes and lymphocytes of Panamanian lizards which showed patent
erythrocytic infections of Plasmodium floridense, Plasmodium tropiduri, and
Plasmodium aurulentum. I suggested two possible interpretations (Telford,
1970): "(1) The exoerythrocytic forms represent one or (probably) more
malaria-like species which parasitize white blood cells exclusively or (2) These
stages are part of the mechanism whereby latent infections can again give rise
to patent parasitemia of the erythrocytes." Scorza (1971) then reported
thrombocytic gametocyte infections in Venezuelan lizards infected with P.
tropiduri. Finding additional infections of malaria-like parasites in
thrombocytes and lymphocytes of Brazilian lizards, Lainson, Landau, and Shaw
(1974) erected a genus, Fallisia, within their family of unpigmented parasites,
the Garniidae, distinguished by supposed absence of an erythrocytic cycle,
schizogony and gametogony occurring solely within thrombocytes and
leucocytes. This position coincided with my first interpretation, cited above.
Their view, apparently, was that saurian malaria species known to parasitize
one series of cells (erythrocytic) lack the capacity to utilize another lineage
(leucocytic). However, pioneer studies by Thompson and Huff (1944)
described the capacity of Plasmodium mexicanum to parasitize virtually all cell
lineages in infections induced by inoculation of blood. In addition, Scorza
(1971) provided substantial evidence that P. tropiduri possesses both
erythrocytic and thrombocytic schizogonic and gametogonic cycles. Until
recently, no evidence has been produced from experimental infections to
preclude the possibility that thrombocytic/lymphocytic infections described as
Fallisia were not preceded by erythrocytic cycles. The usually unpigmented
Plasmodium azurophilum has both erythrocytic and leucocytic cycles of
schizogony and gametogony, for which I suggested (Telford, 1975) that the
non-erythrocytic cycles may represent a defense against immunity raised by a
possibly brief but intense erythrocytic infection. If only leucocytic stages were
available, as is commonly seen in natural infections, one might find it difficult
to distinguish P. azurophilum from Fallisia audaciosa Lainson, Shaw and
Landau, 1975. Experimental evidence is now available, derived from study of
an avian parasite by Gabaldon, Ulloa, and Zerpa (1985), which demonstrates
that the leucocytic cycle of Fallisia neotropicalis is not preceded by an
erythrocytic cycle. Pending confirmation of these results for a saurian Fallisia
species, I can accept Fallisia as a genus of the Plasmodiidae, comprised of
those species for which only thrombocytic or leucocytic cycles are known.

BULLETIN FLORIDA STATE MUSEUM VOL. 34(2)

An additional element of the classical definition of Plasmodium is the
restriction of vector group to mosquitoes. When life cycles of reptilian
plasmodiids were unknown, this was perhaps a useful criterion, being based
upon data from avian and mammalian species only. However, Ayala (1971)
demonstrated that P. mexicanum readily underwent sporogony in two species
of phlebotomine sandflies, Lutzomyia vexator and L. stewarti, and more
recently Petit et al. (1983) obtained the sporogony of Plasmodium agamae in a
European ceratopogonid, Culicoides nubeculosus. Klein (1985) succeeded in
transmitting Plasmodium floridense by Culex erraticus and confirmed Ayala's
work by obtaining transmission of P. mexicanum by bite of infected Lutzomyia
vexator. Ultrastructural studies of P. floridense by Aikawa and Jordan (1968),
P. mexicanum by Moore and Sinden (1973), and P. tropiduri by Scorza (1971)
have demonstrated that there is no significant difference between reptilian and
avian malarial parasites in the morphology of erythrocytic stages, while the
ultrastructure of the sporogonic forms of P. floridense by Klein (1985) and P.
agamae by Boulard et al. (1983) again confirms their generic identity with
Plasmodium. It is appropriate to point out the parallel capacity of adeleid
haemogregarines and lankesterellid coccidia of reptilian hosts to utilize
opportunistically a variety of haematophagous vectors for transmission. This
suggests that reptilian haemosporozoa retain primitive characters which were
of adaptive significance long before vertebrate divergence produced the
reptilian lines which evolved into the birds and mammals of today.
The only components of the classical definition of Plasmodium that are
applicable to all reptilian, avian and mammalian species are the presence of
asexual and sexual cycles in blood cells and tissues of the vertebrate host and
the production of gametocytes. Levine (1985) has re-defined both the family
and the genus in terms broad enough to include all of the known reptilian
haemosporidia: "Macrogamete and microgamont develop independently;
conoid ordinarily absent; syzygy absent; microgamont produces 8 flagellated
microgametes; zygote motile (ookinete); sporozoites naked, with 3-membraned
wall; endodyogeny absent; heteroxenous, with merogony in vertebrate host and
sporogony in invertebrate; pigment (hemozoin) visible with light microscope,
may or may not be formed from host-cell hemoglobin; transmitted by blood-
sucking insects." Although definite proof of an asexual cycle in circulating
leucocytes has not been obtained yet for Saurocytozoon, there is circumstantial
evidence that it exists. I found large intralymphocytic schizonts present during
the first forty days of an initial S. tupinambi infection in a juvenile Tupinambis
teguixin; the schizonts vanished from peripheral blood smears when mature
gametocytes appeared (Telford, 1978). Later (Telford, 1983), I reported
apparent schizonts in lymphocytes of Mabuya multifasciata infected with S.
mabuyi. These observations must be confirmed from experimental infections
induced by sporozoites. Certainly the sporogonic pattern of S. tupinambi, as
described by Landau et al. (1973), justifies its classification as a plasmodiid

TELFORD: CONTRIBUTION TO PLASMODIID SYSTEMATICS

(Telford, 1983). When the two described species are better known, perhaps
Saurocytozoon might be recognized as a subgenus of Plasmodium. At present,
it is reasonable to consider it as a plasmodiid genus (Telford, 1983; Levine,
1985).
As mentioned above, it is desirable to provide subgeneric groupings of
the known reptilian malarias, using criteria similar to those employed by
Garnham (1966) for the definition of mammalian and avian subgenera. He
relied upon two erythrocytic stages to separate the subgenera, gametocyte
shape and schizont size. Sufficient information is known about many of these
parasites to provide other more basic and useful characters for subgeneric
definitions, but they can be grouped by erythrocytic schizonts and gametocytes
alone. With the reptilian malarias we have virtually no useful characters other
than those provided by the erythrocytic stages. I presented some tentative
groupings in 1974, using the range of merozoite mean number and gametocyte
size (length X width, LW), supported by less important characters such as
pigment presence, gametocyte sexual dimorphism, and host cell types. These
groups were based upon study of New World species only, although it was
possible to assign most of the Old World forms to them. Ayala (1977)
provided similar groupings to mine, using also the relationship of parasite size
to host cell nucleus size. Neither of these attempts to group related species
was based upon adequate morphometric comparisons.
In the past decade I have obtained considerably more material, especially
from Africa, but including several species from Southeast Asia as well. The
classification proposed below is based primarily upon morphometric
comparisons, derived from measurements of 17,906 parasites--7213 schizonts
and 10,693 gametocytes, obtained from 316 infections comprised of 115 host-
parasite combinations. There are 67 species and subspecies of Plasmodium
represented, distributed as follows: 6 North American, 19 Middle American,
18 South American, 5 Caribbean, 1 Australian, 4 East-Southeast Asian, and 21
African-Madagascan. Some species (Middle and South American) were
studied from more than one area. Data are included from five species and
subspecies (Africa, Hispaniola, Panama, and the Philippines) presently under
description. These 67 species and subspecies represent 79 percent of those 85
forms known by me to exist.

ACKNOWLEDGEMENTS

Many people have assisted in this work over the last 20 years, most with no expectation of
significant results, for progress has been slow. The preparation and screening of blood smears
from over 15,000 lizards, the study of infections found, the measurement of series of parasites,
their frequent taxonomic description accompanied by the taking of photomicrographs and
drawings, seemingly endless analysis of data, and evaluation of combinations of characters have

BULLETIN FLORIDA STATE MUSEUM VOL. 34(2)

all consumed my time and energy. Very little of this work could be done during the normal
working day, for I have never been fortunate enough to be employed as a student of either
malarial parasites or their saurian hosts. My professional obligations to the Gorgas Memorial
Laboratory, the University of Florida, the World Health Organization, and the Danish
International Development Agency have held first priority, although when time permitted it
certainly must have appeared to my superiors that my interest in saurian malaria came first!
Hopefully my productivity on the various projects for which I was engaged by those agencies
reduced to some degree the anxiety of my superiors. Only the most efficient use of personal time
made it possible to produce the body of work which resulted in this contribution.
I wish to thank the following individuals for their help in the acquisition of the lizard hosts
or blood slides: the late Howard W. ("Duke") Campbell, Richard C. Goris, Walter Auffenberg,
Richard Franz, Steven P. Christman, Peter Meylan, John B. Iverson, C. A. Smith, Paul Moler,
Sylvia Scudder, David G. Young, Stephen C. Ayala, J. V. Scorza, John E. Lovett, William R.
Smythe, Kim Howell, John Hall, Richard D. Sage, G. Gorman, A. Lowichik, T. A. Burns, and C.
H. Lowe. A very distinguished group of senior scientists through the years have provided the
very best professional knowledge or contributed blood slides, furnished administrative support,
and served as role models through their interest, friendship, and above all else, constructive
criticism, and to them I am deeply indebted: P. C. C Garnham, Martin D. Young, Manabu Sasa,
the late Gordon H. Ball, the late L. A. Stauber, and the late C. G. Huff. I wish to thank Jerry F.
Butler for his administrative support toward publication of this study, during a most difficult
transition period for me. I have had the pride and joy of watching three small boys grow into
manhood enthusiastically catching every lizard sighted as we explored together the rice fields of
Japan, the deserts and mountains of the American southwest and Pakistan, the tropical forests of
Panama and Venezuela, the urban parks of Southeast Asia, the East African savanna and
montane rainforest, and the woods, swamps and neighborhoods of North Florida. Sam,
Randolph, and Robert collected the hosts of probably one-third of the malaria species I
subsequently described as new, and their assistance is missed as their own careers develop. My
wife, Michiko, has helped me in countless ways during three decades, providing the domestic
stability without which this work could not have been done.

MATERIALS AND METHODS

I have elsewhere (Telford, 1974, 1979) described 21 characters of possible taxonomic utility
in defining saurian Plasmodium species. Most of them await proper statistical evaluation,
because the very large number of parasites examined precludes their use until all data have been
properly prepared for multivariate analysis by computer. I chose five characters to consider for
definition of subgenera: size of gametocyte and schizont (maximum length X maximum width, or
LW), merozoite number, gametocyte shape (length/width, or L/W), and gametocyte-schizont size
relative to erythrocyte nucleus size. I have discarded gametocyte shape in the present analysis.
Although useful in the definition of many species, where 95 percent of gametocytes may show a
characteristic form (elongate, oval or round, bulky), in other species there is a clear correlation
between gametocyte shape and the position of the gametocyte within the host cell. In the most
euryxenous saurian Plasmodium, for instance P. floridense, gametocyte shape is significantly
correlated (by chi square test) with position: those gametocytes with an L/W ratio of 1.7+ tend
to occupy lateral or lateropolar positions, while those with round (1.0-1.39) or oval (1.4-1.69)
configurations occupy polar positions. The comparison was run on 1318 gametocytes from
infections of 11 Anolis and 2 Sceloporus species. Schizont size is correlated with merozoite
numbers in some species but not in others, but I have decided to use this characteristic in
subgeneric definitions because it is easily compared with erythrocyte nucleus size. As with
gametocyte shape, the configuration of saurian Plasmodium schizonts is too variable in most
species to permit simple generalizations to describe shape, and thus was not used here for
subgeneric definitions.

TELFORD: CONTRIBUTION TO PLASMODIID SYSTEMATICS

A simple comparison of gametocyte or schizont size with the nucleus of the parasitized cell
introduces other variables into this relationship: the effect the parasite has upon the host cell
nucleus. Many species do cause hypertrophy, hypotrophy, or mild to severe distortion and even
lysis of the host cell nucleus. While nuclei of mature erythrocytes vary within comparable limits
to that seen for gametocytes and schizonts, nuclei of erythroblasts and proerythrocytes are far
more variable, both in shape and size. Immature cells are often parasitized by asexual stages in
particular, and in some cases, preferentially. It was desirable, therefore, to provide an objective
standard against which size comparisons could be made, one always available and subject to the
same techniques of fixation, staining and measurements as the parasites themselves, yet
independent from possible effects of parasitism. The size (LW) of nuclei from uninfected,
mature erythrocytes provides this standard. By using samples of nuclei from the slides examined,
the significant differences that exist in erythrocyte nucleus size among host families, genera and
species groups can be disregarded. Accordingly, I have plotted ratios of mean schizont size to
mean erythrocyte nucleus size against ratios of mean gametocyte size to the same nucleus mean
size for each infection studied. Both ratios have been plotted against the range of merozoite
numbers observed in the same infection. Mean merozoite number is too susceptible to the
influence of infection phase, commonly being lower following the infection peak of parasitemia.
The range of merozoite numbers is much more similar during each phase of infection (early, pre-
peak, post-peak, chronic, and recrudescent or relapse phases) than is the mean in most of the
species I have studied. The point chosen for plotting against the schizont and gametocyte size
ratios is the midpoint of the observed range. The location of a sample within the triangle formed
by these three characters provides a locus which can be considered to be the phenotype of the
given species as defined by these characters. Incorporation of additional characters would, of
course, change the locus but, hopefully, not the groupings of the species.
Additional considerations have influenced the graphical presentation of the data. For
simplicity in presentation, all means used and the range midpoint of merozoites have been
converted to log values. Of the 67 species and subspecies studied, one (Plasmodium maculilabre)
was represented only by gametocytes, no mature schizonts being present on the slides examined.
These could not be plotted. The plotting of individual infections measured-over 300-found
many points representing the individual infections overlapping, both within species of which
several infections were measured and among species. However, if an average of the mean values
or merozoite range midpoints is used, most of the overlap disappears, and species can be
represented upon the graph by a single number, that associated with the host-parasite association
listed under Material Examined. Morphometric data have not been used to define two
subgenera recognized here, Garnia and Ophidiella, and they are therefore not included in the
figures.
All measurements were taken by me with the same microscope (a Nikon STR) and the
same ocular micrometer, calibrated at 1000X, under oil immersion. The usual samples taken
were 25 gametocytes, 25 mature or segmenting schizonts, and 10 uninfected erythrocytes. With
undescribed species, species where few infections could be obtained, or when infections were
intense, thus reducing the time involved to locate parasites, sample size was often increased to 50
or more of each stage. When possible, three infections from each host-parasite association were
studied, and in some cases more. Some, however, are represented by only a single infection
(36%), and not all samples of each stage were adequate for plotting. Selection of parasites for
measurement was as unbiased as possible: the first seen which showed the following criteria were
measured, and each subsequent parasite until the desired sample had been found. Gametocytes
were chosen on the basis of apparent maturity: sexual differentiation by staining reaction, and
dispersal of pigment from the clumped mass seen in immature gametocytes of virtually all species
studied. Those few species that show focused pigment when apparently mature were chosen by
comparison of staining reaction and size to obviously immature gametocytes. Schizonts selected
for measurement were either segmenting or those in which nuclear division had evidently been
completed, but discrete merozoites were not visible. Use of this latter category cannot be
defended except by pleading experience of the investigator. For gametocytes and schizonts,
maximum length and maximum width were measured, which provided the character length X
width (LW). Schizonts were selected for measurements only if the number of merozoites or
nuclei could be counted. Uninfected erythrocytes from each infection studied were those which
showed no distortion from the act of smearing the blood on the slide, or from lysis. Maximum

length and width was recorded for both the cell and its nucleus. Cell dimensions were also
routinely recorded for those mature erythrocytic cells containing measured parasites, as were the
other characters described earlier (Telford, 1974, 1979). All slides examined had been fixed in
absolute methanol and stained by Giemsa or May-Grunwald Giemsa techniques.

RESULTS

In Figure 1 multiple samples from four saurian species host to
Plasmodium oridense have been plotted around the mean of all samples from
the

TELFORD: CONTRIBUTION TO PLASMODIID SYSTEMATICS

!

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log x gametocyte LW/ x rbc nucleus LW
Figure 2. Separation of saurian Plasmodium species (from both Eastern and Western
Hemispheres) by means of morphometric characters plotted against midpoint of the observed
merozoite range. Sample numbers refer to each host-parasite association listed under Material
Examined. Samples 1-10 are species with large gametocytes and schizonts; 11-24 have small
gametocytes and schizonts; 82-85 have large gametocytes and medium sized schizonts; 86-88 have
schizonts disproportionately smaller than gametocytes.

particular hosts. The observed variation found in the samples from a given
host-parasite association lies within certain limits around the point
representing the overall average. It is probable that this variation can be
attributed largely to infection phase which can influence gametocyte or
schizont size, or merozoite range. Figure 2 demonstrates the presence of two
groups of Plasmodium species widely separated by size of both gametocytes
and schizonts: very large and very small. The group of larger species contains
within it Plasmodium diploglossi Aragao & Neiva, 1909, type species of the

BULLETIN FLORIDA STATE MUSEUM VOL. 34(2)

C.,

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q D
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log x gametocyte LW/ x rbc nucleus LW

Figure 3. Samples from Neotropical Plasmodium species that have gametocytes and schizonts
of similar size, intermediate between Sauramoeba and Carinamoeba species.

subgenus Sauramoeba Garnham, 1966. The smallest species include
Plasmodium minasense Carini & Rudolph, 1912, type species of Carinamoeba
Garnham, 1966. I recognize these subgenera as valid groups of
morphometrically related species.
There exists, however, a very large number of species that lie in between
Sauramoeba and Carinamoeba in their morphometric characters (Figs. 3-6).
They are cleanly separated from Sauramoeba (Fig. 7). Some species, however,
do overlap with Carinamoeba through the influence, usually, of having smaller
schizonts which contain, however, more merozoites than typically seen in
Carinamoeba species (Fig. 7). The midpoint in range of merozoite number for
Carinamoeba is less than 8, i.e. 4-10 merozoites are produced by three normal

5,
5,

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TELFORD: CONTRIBUTION TO PLASMODIID SYSTEMATICS

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log x gametocyte LW/ x rbc nucleus LW

Figure 4. Samples from Old World Plasmodium species that have gametocytes and schizonts
of similar size, intermediate between Sauramoeba and Carinamoeba species.

nuclear divisions, with occasional nuclei undergoing an extra division; this
produces a greater effect upon midpoint of range than upon mean. The
intermediate sized species undergo four or five nuclear divisions, with a range
midpoint of 8 or more, i.e. 4-12+. Plasmodium basilisci Pelaez & Perez-
Reyes, 1959 is a case in point. In the type host, Basiliscus vittatus, it produces
4-8 merozoites, which would place it in Carinamoeba. Infections in Basiliscus
basiliscus usually show the same range in numbers but when immature
erythrocytes are parasitized, up to 14 merozoites can be found (Telford, 1972).
Its relationships morphometrically, then exclude it from Carinamoeba.
If the intermediate sized species from both Western (Fig. 3) and Eastern
(Fig. 4) Hemispheres are plotted together (Figs. 5, 6), the combined polygon,
formed by means of the various host-parasite associations, contains within it

104 (76 %) of the 137 individual samples (Fig. 6). Enough material from both
Africa and the Western Hemisphere has been examined to demonstrate that
the same three basic groupings of large, intermediate, and small species occur
in both areas (Figs. 2, 5). I believe that this justifies recognition of a third
subgenus of pigmented Plasmodium species from lizards, intermediate in size
between Sauramoeba and Carinamoeba. It is described below as subgenus
Lacertamoeba.
Four additional subgenera can be recognized at present. One of these is
Gamia Lainson, Landau and Shaw, 1971, to which I assign all unpigmented
erythrocytic species in which the absence of pigment is not related to
parasitization of immature erythrocytic cells. If Gamia were to be plotted in
Figure 7 with the pigmented subgenera, one species, Plasmodium balli, would

TELFORD: CONTRIBUTION TO PLASMODIID SYSTEMATICS

a

4
3

uCi
d
-j

log 7 gametocyte LW/ R rbc nucleus LW

Figure 6. Distribution of individual Neotropical and Old World samples (solid circles) in
relation to the polygon representing the subgenus Lacertamoeba. Numbered circles represent
species that form the polygon boundary within which all other species means are included.

clearly lie within Sauramoeba and the remaining eight species within
Lacertamoeba, which suggests that pigment presence or absence may be a
derived rather than basic character. Until the significance of pigment presence
or absence and its variability within individual infections is better understood,
the definition provided here for Gamia must suffice. Although I have recently
considered Fallisia to be a subgenus of Plasmodium (Telford, 1986), in
Levine's classification (1985) Fallisia was synonymized with Plasmodium. The
several species, though, evidently lack an erythrocytic cycle, with both
schizogony and gametogony taking place in thrombocytes and leucocytes. It is
possible that another explanation may yet be found for this choice of host cell,
but on present knowledge, in view of the findings of Gabaldon et al. (1985), it

is reasonable to recognize Fallisia as a genus of the Plasmodiidae, as stated
above. Garnham (1966) erected Ophidiella as a subgenus to accommodate
Plasmodium species of snakes. Ophidiella is too poorly known for critical
definition: there are three described species, one of which (Plasmodium
tomodoni) could represent an ophidian member of Sauramoeba, and two of
which (Plasmodium wenyoni and Plasmodium pessoai) might be Lacertamoeba
species. Until more data are available, therefore, I will follow Garnham (1966)
in recognizing the subgenus as described, despite my reservations on the
significance of host type in systematics. Two North American species,
Plasmodium mexicanum and P. chiricahuae, do not fit well morphometrically

TELFORD: CONTRIBUTION TO PLASMODIID SYSTEMATICS

with Lacertamoeba species (Fig. 7). Their gametocytes are among the largest
known, yet schizonts are of medium size. In P. mexicanum it is possible to
produce exoerythrocytic schizonts in a variety of fixed and circulating cells by
inoculation of infected blood into a clean host (Thompson and Huff, 1944a).
Pathology can result from apparent occlusion of cerebral capillaries by these
exoerythrocytic schizonts. This has never been reported for the Lacertamoeba
species which have been studied in experimental infections: P. tropiduri
(Scorza, 1970), P. floridense (Thompson and Huff, 1944b; Goodwin and
Stapleton, 1955), and P. sasai (Telford, 1972). In addition, sporogony takes
place in psychodid flies (Ayala, 1971; Klein, 1985), and these probably are the
natural vectors (Ayala, 1973). Therefore, it is appropriate to place these two
North American species into a separate subgenus which I designate
Paraplasmodium. The poorly known Venezuelan species, Plasmodium pifanoi
Scorza & Dagert, 1956 has very large gametocytes and medium sized schizonts,
and on these grounds only can be included in this subgenus. Finally, there
appears to be an Asian-Pacific group of pigmented species, still poorly known,
which has the unlikely combination of disproportionately large gametocytes
and tiny schizonts. Plasmodium saurocaudatum Telford, 1983, P. lygosomae
Laird,1951, P. vastator Laird,1960, and Plasmodium clelandi Manawadu, 1972
can be placed in this subgenus, Asiamoeba.

Definition of Subgenera

Sauramoeba Garnham, 1966

Saurian Plasmodium species characterized by large schizonts and
gametocytes. Mean schizont size is three to seven times that of uninfected
erythrocyte nuclei. Schizonts undergo 4 to 7 nuclear divisions, producing 14-
130 merozoites. Mean gametocyte size is two to five times that of nuclei from
uninfected erythrocytes. Gametocytes are usually smaller than or equal to
schizonts in size. Sexual dimorphism is usually present in gametocyte size:
macrogametocytes are larger than microgametocytes. Gametogony occurs in
the erythrocytic series. Pigment is always present. Secondary erythrocytic
schizogony occurs in leucocytes. Sporogony is unknown.

Saurian Plasmodium species characterized by small schizonts and
gametocytes. Schizont size averages smaller than that of uninfected
erythrocyte nuclei. Schizonts undergo 2 or 3 nuclear divisions, typically
producing 4-8 merozoites, rarely up to 12. Average gametocyte size may
slightly exceed that of uninfected erythrocyte nuclei, but is usually somewhat
less. Gametocyte size may be twice that of schizonts. Sexual dimorphism in
gametocyte size may occur but is not characteristic of most species. Pigment is
always present in larger asexual stages and gametocytes. Gametogony occurs
only in the erythrocytic series, usually in mature cells. Secondary
exoerythrocytic schizonts may parasitize thrombocytes. Sporogony is unknown.

Saurian Plasmodium species characterized by medium sized schizonts
and gametocytes. Mean schizont size is one-half to twice that of nuclei from
uninfected erythrocytes. Schizonts typically undergo 3 to 5 nuclear divisions; 4-
55 merozoites may be produced. Average gametocyte size varies from slightly
less than that of uninfected erythrocyte nuclei to twice their size. Gametocytes
may be two and one-half times larger than schizonts, but more commonly
slightly exceed schizonts in size. Pigment is usually visible; its absence is
correlated with immaturity of host cells. Gametogony occurs in erythrocytes,
but in some species thrombocytes or lymphocytes may host sexual stages.
There is no consistent sexual difference in size of gametocytes. Where known,
exoerythrocytic schizogony occurs in the reticulo-endothelial system, notably in
the lymphoid macrophage series. Sporogony can occur in culicid and
ceratopogonid flies.

Saurian Plasmodium species characterized by medium sized schizonts
and large gametocytes. Mean schizont size is one-half to twice that of
uninfected erythrocyte nuclei. Schizonts undergo 3 to 5 nuclear divisions,
producing 4-30 merozoites. Mean gametoctye size is three to six times that of
nuclei from uninfected erythrocytes. Gametocytes are three to six times the
size of schizonts. Pigment is always present in erythrocytic parasites.
Gametogony occurs in erythrocytes. Sexual dimorphism is present in
gametocyte size: macrogametocytes are larger than microgametocytes.
Secondary exoerythrocytic schizonts may occur in fixed cells of the viscera and
in circulating white blood cells. Sporogony can occur in psychodid flies.

Saurian Plasmodium species characterized by schizonts and gametocytes
greatly disproportionate in size. Schizont size does not exceed one-fourth that
of nuclei from uninfected cells. Schizonts undergo 2 nuclear divisions,
producing 3-4 nuclei. Mean gametocyte size is four to eight times that of
uninfected erythrocyte nuclei. Gametocytes are four to fifteen times larger
than schizonts. Pigment is always present. Gametogony occurs in
erythrocytes. Sexual dimorphism may be present in gametocyte size, with
macrogametocytes larger than microgametocytes. Exoerythrocytic schizogony
and sporogony are unknown.

Saurian Plasmodium species characterized by erythrocytic schizonts and
gametocytes equal to or larger than uninfected erythrocyte nuclei, in which the
absence of pigment is independent of host cell maturity. Pigment is rarely
demonstrable. Schizonts undergo 3 to 7 nuclear divisions, producing 8 to 100
merozoites. Gametogony may occur in erythrocytes or in leucocytes during
part of the life cycle. Gametocytes may show sexual dimorphism in size, with
macrogametocytes usually larger than microgametocytes. Exoerythrocytic
schizonts occur in thrombocytes and leucocytes. Sporogony is unknown.

The subgeneric classification presented above for the reptilian
haemosporidia which I consider to be Plasmodium is open to criticism from at
least three aspects. Those workers who insist that Plasmodium should include
only those organisms which share all characters shown by the four species that
parasitize humans--specifically the presence of pigment, restriction to
erythrocytes in the vertebrate, and use of the Culicidae as vectors--will be

TELFORD: CONTRIBUTION TO PLASMODIID SYSTEMATICS

reluctant to accept the scheme. In a zoological, as opposed to historical, sense
there is nothing special about the fact that the genus Plasmodium Marchiafava
and Celli, 1885 was described from a species, P. malariae, which infects
humans. Instead, I take the position that classifications, like species, are not
immutable, and while I readily recognize new species on the basis of
morphometric and qualitative traits, I prefer to conceive of the higher taxa of
genus and family as showing probable evolutionary relationships. It is most
unusual to demonstrate a derivative/phylogenetic relationship of one species
group to another currently existing, except in a speculative manner based upon
the experience of the systematist involved. Happily, with the advent of
biochemical techniques that demonstrate actual sharing of portions of
genomes, it should become possible to establish relationships in more objective
terms than was previously possible for organisms which lack a fossil record.
Another anticipated criticism is my use of morphometrics as the basis for
the classification. I can only respond that there is little else available yet for
parasites of lower vertebrates besides those characters which can be gleaned
from blood smears. Professor Garnham (1966) provided adequate justification
for this approach to taxonomic characters: "The organisms are so small that
what would constitute familial or even ordinal differences of the same
proportions in larger animals, could easily pass unrecognized in the Protozoa;
any character therefore should be seized and used as liberally as possible."
The third possible objection will be that there are too many subgenera of
reptilian plasmodiids in comparison to mammals and birds. Garnham (1966)
proposed three subgenera for mammalian parasites: Plasmodium, Laverania,
and Vinckeia. Avian Plasmodium species were placed in four: Haemamoeba,
Huffia, Novyella, and Giovannolaia. Here, I suggest that there are seven
groups of possibly related species found in reptilian hosts: Sauramoeba,
Carinamoeba, Lacertamoeba, Paraplasmodium, Asiamoeba, Gamia, and
Ophidiella. Fallisia and Saurocytozoon deserve generic status, which is a
different problem. Given the fact that there are now as many described and
known but yet undescribed species and subspecies of reptilian plasmodiids as
there are avian and mammalian species together, I do not think there is an
excessive number of subgenera. Plasmodiids have been reptilian parasites far
longer than they have utilized mammalian and avian hosts, and increased
diversity of both species and species groups is to be expected.
The topics of variation and ecological zoogeography will not be discussed
here in detail, as I intend to address them in future papers. It is obvious,
though, from the information presented here, that not all of the reptilian
genera and subgenera are cosmopolitan in their distribution. To a
considerable degree this represents the geographical distribution and intensity
of investigative effort, which has been greatest in the Western Hemisphere and
East Africa. The distribution of subgenera and genera can be briefly
summarized as follows: Sauramoeba is known from Mexico to Brazil,

BULLETIN FLORIDA STATE MUSEUM VOL. 34(2)

throughout Africa and on Madagascar, and from Australia, but not yet from
Asia; Carinamoeba occurs from Mexico through Brazil, in the Caribbean,
Africa, and Southeast Asia; Lacertamoeba is found throughout the Western
Hemisphere, Africa, Madagascar, Southeast and East Asia, and Australia;
Gamia, Ophidiella, and Paraplasmodium are known only from the Western
Hemisphere; Asiamoeba is found in Southeast Asia and New Zealand; Fallisia
occurs in the Neotropics from Brazil at least into Panama, in the Caribbean,
and in Southeast Asia and the Australasian region; Saurocytozoon is found in
northern South America and in Southeast Asia. Three important areas are yet
poorly known--Southeast Asia, Australasia, and the West and Central African
rainforest. Sustained field efforts in these areas should clarify the distribution
of reptilian haemosporidia and hopefully shed additional light upon their
relationships. The classification provided here should be tested by studies on
sporogony and exoerythrocytic schizogony and by biochemical procedures to
determine the relevance of morphometrics to systematics of the
Haemosporidia.

The material listed below was examined during the period 1967-86. Except where noted,
all slides are in the Telford collection or have been deposited as paratype material as indicated in
earlier taxonomic papers. Voucher specimens of hosts for all material I have collected is
deposited at the Florida State Museum, Gainesville. Unless another source is indicated, I have
personally obtained the hosts and prepared the slides. Numbers preceding the taxonomic name
identify the species or samples in the figures. The letters S and G refer to the stages measured,
schizont and gametocyte, respectively, while N and E indicate type of infection, natural or
experimental. Host species immediately follows the Plasmodium specific name. TYPE indicates
that I have examined the type slide.

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